EP3870331B1 - Modifizierung der antikörper-fcrn-bindung - Google Patents

Modifizierung der antikörper-fcrn-bindung

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Publication number
EP3870331B1
EP3870331B1 EP19795164.3A EP19795164A EP3870331B1 EP 3870331 B1 EP3870331 B1 EP 3870331B1 EP 19795164 A EP19795164 A EP 19795164A EP 3870331 B1 EP3870331 B1 EP 3870331B1
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EP
European Patent Office
Prior art keywords
antibody
fcrn
retention time
amino acid
affinity chromatography
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EP19795164.3A
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English (en)
French (fr)
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EP3870331C0 (de
EP3870331A1 (de
Inventor
Thomas Emrich
Hubert Kettenberger
Thomas Kraft
Wolfgang Richter
Tilman Schlothauer
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F Hoffmann La Roche AG
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F Hoffmann La Roche AG
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B01D15/3809—Affinity chromatography of the antigen-antibody type, e.g. protein A, G or L chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/524—CH2 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/72—Increased effector function due to an Fc-modification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the neonatal Fc receptor FcRn is a membrane-associated receptor involved in both IgG and albumin homeostasis, in maternal IgG transport across the placenta and in antigen-IgG immune complex phagocytosis (see, e.g., Brambell, F.W., et al., Nature 203 (1964) 1352-1354 ; Ropeenian, D.C., et al., J. Immunol. 170 (2003) 3528-3533 ).
  • Human FcRn is a heterodimer consisting of the glycosylated class I major histocompatibility complex-like protein ( ⁇ -FcRn) and a ⁇ 2 microglobulin ( ⁇ 2 m) subunit (see, e.g., Kuo, T.T., et al., J. Clin. Immunol. 30 (2010) 777-789 ).
  • FcRn binds to a site in the C H 2-C H 3 region of the Fc-region (see, e.g., Ropeenian, D.C. and Akilesh, S., Nat. Rev. Immunol. 7 (2007) 715-725 ; Martin, W.L., et al., Mol.
  • the affinity between the FcRn and the Fc-region is pH dependent, showing nanomolar affinity at endosomal pH of 5-6 and rather weak binding at a physiological pH of 7.4 (see, e.g., Goebl, N.A., et al., Mol. Biol. Cell 19 (2008) 5490-5505 ; Ober, R.J., et al., Proc. Natl. Acad. Sci. USA 101 (2004) 11076-11081 ; Ober, R.J., et al., J. Immunol. 172 (2004) 2021-2029 ).
  • the underlying mechanism conveying long half-life to IgGs can be explained by three fundamental steps.
  • IgGs are subject to unspecific pinocytosis by various cell types (see, e.g., Akilesh, S., et al., J. Immunol. 179 (2007) 4580-4588 ; Montoyo, H.P., et al., Proc. Natl. Acad. Sci. USA 106 (2009) 2788-2793 .).
  • IgGs encounter and bind FcRn in the acidic endosome at a pH of 5-6, thereby protecting IgGs from lysosomal degradation (see, e.g., Ropeenian, D.C. and Akilesh, S., Nat. Rev. Immunol.
  • IgGs are released in the extracellular space at physiological pH of 7.4 (see, e.g., Ghetie, V. and Ward, E.S., Annu. Rev. Immunol. 18 (2000) 739-766 ).
  • This strict pH-dependent bind-and-release mechanism is critical for IgG recycling and any deviation of the binding characteristics at different pH values may strongly influence circulation half-life of IgGs (see, e.g., Vaccaro, C., et al., Nat. Biotechnol. 23 (2005) 1283-1288 ).
  • Hoetzel, I., et al. disclosed a strategy for risk mitigation of antibodies with fast clearance as a majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. It is described an assay based on ELISA detection of binding to baculovirus (BV) particles to evaluate the non-specific binding of therapeutic proteins.
  • BV baculovirus
  • WO 2015/140126 discloses a method for the prediction of the in vivo half-life of an antibody based on the retention time determined on an FcRn affinity chromatography column.
  • FcRn chromatography see, e.g., Schoch, A., et al., Proc. Natl. Acad. Sci. USA 112 (2015) 5997-6002
  • FcRn affinity see, e.g., Neuber, T., et al., MAbs 6 (2014) 928-942
  • heparin binding e.g. in ELISA format (see, e.g., Datta-Mannan, A., et al., MAbs 7 (2015) 1084-1093 ) is disclosed as a surrogate parameter to quantify non-specific interactions with cell surface structures.
  • WO 2015/189249 discloses a method for selecting antibodies with modified FcRn interaction.
  • Fc-receptor binding modified asymmetric antibodies and methods of use are disclosed in US 2016/019489 .
  • US 2017/227547 the in vitro prediction of in vivo half-life is disclosed.
  • Antibodies binding to STEAP-1 are disclosed in WO 2018/184966 .
  • the method according to the invention is based on the combination of two different chromatographic methods, i.e. the combination of FcRn affinity chromatography and heparin affinity chromatography.
  • the combination of FcRn affinity chromatography and heparin affinity chromatography allows defining FcRn and heparin affinity chromatography column retention time thresholds and thereby a two-dimensional retention time region, wherein antibodies with slow clearance, i.e. long systemic circulation half-live, can be found.
  • an increase of the retention time in an FcRn affinity chromatography by 1 minute or more but less than about 5 minutes provides for the most efficient modification with respect to improving the in vivo half-live.
  • the increase is more than 3 minutes but less than 4 minutes.
  • a method for providing a modified antibody with improved in vivo half-live comprising the modification of the Fc-region of the antibody by introducing one or more mutations that change the binding of the Fc-region to human FcRn until the (relative) retention time of the modified antibody in an FcRn affinity chromatography is increased for more than 1 minute but not more than 5 minutes compared to the parent antibody.
  • one aspect of the current invention is a method for providing an antibody with improved in vivo half-live, comprising the following steps:
  • one aspect of the current invention is a method for providing an antibody with improved in vivo half-live, comprising the following steps:
  • the change of the relative retention time between the (parent) antibody and the modified antibody is more than about 2 minute and less than about 4.5 minutes.
  • the change of the relative retention time between the (parent) antibody and the modified antibody is more than about 3 minute and less than about 4 minutes.
  • the change of the relative retention time between the (parent) antibody and the modified antibody is more than about 3.3 minute and less than about 3.9 minutes.
  • the anti-Her3 antibody has a heavy chain of SEQ ID NO: 03 and a light chain of SEQ ID NO: 04.
  • the FcRn is a non-covalent complex with human beta-2-microglobulin (b2m).
  • the method comprises as step b) the following step: b) modifying the Fc-region of the antibody by introducing mutations that increase the binding of the Fc-region to human FcRn to obtain a modified antibody, and modifying the Fv of the antibody by
  • a relative retention time region comprising predominantly antibodies with slow clearance is defined. This region is defined by a relative retention time on the FcRn affinity chromatography column of less than 1.78 (with an oxidized (H2O2-treated) anti-Her3 antibody preparation as reference antibody) and by a relative retention time on the heparin affinity chromatography column of less than 0.87 (with an anti-pTau antibody as reference antibody).
  • the modified antibody has at least one additional negatively charged patch on its (solvent-exposed) surface.
  • the modified antibody has the same (surface) net charge as the parent antibody.
  • a (permanently) negatively charged amino acid residue is selected from the group consisting of glutamate and aspartate.
  • a (permanently) positively charged amino acid residue is selected from the group consisting of arginine and lysine.
  • the pH-dependently charged amino acid residue is histidine.
  • a permanently charged amino acid residue has the same (net) charge in the pH range from pH 6 to pH 8.
  • a pH-dependently charged amino acid residue has a first (net) charge at pH 6 and an opposite second (net) charge at pH 8.
  • the standard sample is an oxidized antibody preparation comprising the reference antibody with respect to the methionine residues at position 252 in the heavy chain CH2 domains in non-oxidized form, in mono-oxidized form (only one of the two methionins at position 252 is oxidized) and in bi-oxidized form (both methionine residues at position 252 are oxidized) (numbering according to Kabat).
  • the first threshold value in step f) is 2. In one embodiment the first threshold value is 1.8. In one embodiment the first threshold value is 1.78.
  • the second reference antibody in step f) is an anti-pTau antibody that has a heavy chain with the amino acid sequence of SEQ ID NO: 01 and a light chain with the amino acid sequence of SEQ ID NO: 02.
  • the second threshold value is 1. In one embodiment the second threshold value is 0.8. In one embodiment the second threshold value is 0.78.
  • step f) is providing an antibody with improved in vivo half-live if the modified antibody has
  • beta-2-microglobulin is from the same species as the FcRn.
  • beta-2-microglobulin is from a different species as the FcRn.
  • the reference antibody for the FcRn affinity chromatography is the anti-HER3 antibody with SEQ ID NO: 03 (heavy chain) and SEQ ID NO: 04 (light chain).
  • the reference antibody for the heparin affinity chromatography is the anti-pTau antibody with SEQ ID NO: 01 (heavy chain) and SEQ ID NO: 02 (light chain).
  • the antibody is a monospecific antibody or antibody fragment of fusion polypeptide, or a bispecific antibody or antibody fragment of fusion polypeptide, or a trispecific antibody or antibody fragment of fusion polypeptide, or a tetraspecific antibody or antibody fragment of fusion polypeptide.
  • the antibody is an antibody of the class IgG. In one embodiment the antibody is an antibody of the subclass IgG1, IgG2, IgG3 or IgG4. In one embodiment the antibody is an antibody of the subclass IgG1 or IgG4.
  • the invention is based, at least in part, on the finding that for the improvement of in vivo pharmacokinetic properties a defined increase of FcRn binding as determined by affinity chromatography on an FcRn affinity column is advantageous.
  • FcRn affinity chromatography and heparin affinity chromatography allows to define FcRn and heparin affinity chromatography column retention time thresholds and thereby a retention time region, wherein antibodies with slow clearance, i.e. long systemic circulation half-live, can be found.
  • retention times on an FcRn affinity chromatography column and on a heparin affinity chromatography column are normalized based on the retention times of reference antibodies on the respective columns a relative retention time region comprising predominantly antibodies with slow clearance is defined.
  • This region is defined by a relative retention time on the FcRn affinity chromatography column of less than 1.78 (with an anti-Her3 antibody as reference antibody) and by a relative retention time on the heparin affinity chromatography column of less than 0.87 (with an anti-pTau antibody as reference antibody).
  • amino acid positions of all constant regions and domains of the heavy and light chain are numbered according to the Kabat numbering system described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991 ) and is referred to as "numbering according to Kabat" herein.
  • Kabat numbering system see pages 647-660 of Kabat, et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991 ) is used for the light chain constant domain CL of kappa and lambda isotype
  • Kabat EU index numbering system see pages 661-723 is used for the constant heavy chain domains (CH1, Hinge, CH2 and CH3, which is herein further clarified by referring to "numbering according to Kabat EU index" in this case).
  • knobs into holes dimerization modules and their use in antibody engineering are described in Carter P.; Ridgway J.B.B.; Presta L.G.: Immunotechnology, Volume 2, Number 1, February 1996, pp. 73-73 (1).
  • the use of recombinant DNA technology enables the generation derivatives of a nucleic acid.
  • Such derivatives can, for example, be modified in individual or several nucleotide positions by substitution, alteration, exchange, deletion or insertion.
  • the modification or derivatization can, for example, be carried out by means of site directed mutagenesis.
  • Such modifications can easily be carried out by a person skilled in the art (see e.g. Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA ; Hames, B.D., and Higgins, S.G., Nucleic acid hybridization - a practical approach (1985) IRL Press, Oxford, Engl and).
  • the term "about” denotes a range of +/- 20 % of the thereafter following numerical value. In one embodiment the term about denotes a range of +/- 10 % of the thereafter following numerical value. In one embodiment the term about denotes a range of +/- 5 % of the thereafter following numerical value.
  • antibody herein is used in a broad sense and encompasses various antibody structures, including but not limited to monoclonal full length antibodies and multispecific antibodies (e.g. bispecific antibodies, trispecific antibodies) so long as they have an Fc-region.
  • multispecific antibody denotes an antibody that has binding specificities for at least two different epitopes on the same antigen or two different antigens.
  • Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g. F(ab')2 bispecific antibodies) or combinations thereof (e.g. full length antibody plus additional scFv or Fab fragments).
  • Engineered antibodies with two, three or more (e.g. four) functional antigen binding sites have also been reported (see, e.g., US 2002/0004587 A1 ).
  • binding denotes the binding of an antibody in an in vitro assay. In one embodiment binding is determined in a binding assay in which the antibody is bound to a surface and binding of the antigen to the antibody is measured by Surface Plasmon Resonance (SPR). Binding means e.g. a binding affinity (K D ) of 10 -8 M or less, in some embodiments of 10 -13 to 10 -8 M, in some embodiments of 10 -13 to 10 -9 M. The term “binding” also includes the term “specifically binding”.
  • buffer substance denotes a substance that when in solution can level changes of the pH value of the solution e.g. due to the addition or release of acidic or basic substances.
  • the "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • Fc-fusion polypeptide denotes a fusion of a binding domain (e.g. an antigen binding domain such as a single chain antibody, or a polypeptide such as a ligand of a receptor) with an antibody Fc-region.
  • a binding domain e.g. an antigen binding domain such as a single chain antibody, or a polypeptide such as a ligand of a receptor
  • Fc-region of human origin denotes the C-terminal region of an immunoglobulin heavy chain of human origin that contains at least a part of the hinge region, the CH2 domain and the CH3 domain.
  • a human IgG heavy chain Fc-region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain.
  • the Fc-region has the amino acid sequence of SEQ ID NO: 05.
  • the C-terminal lysine (Lys447) of the Fc-region may or may not be present.
  • the Fc-region is composed of two heavy chain Fc-region polypeptides, which can be covalently linked to each other via the hinge region cysteine residues forming inter-chain disulfide bonds.
  • FcRn denotes the human neonatal Fc-receptor. FcRn functions to salvage IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life.
  • the FcRn is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein ( ⁇ -FcRn) and a 15 kDa ⁇ 2-microglobulin ( ⁇ 2m). FcRn binds with high affinity to the CH2-CH3 portion of the Fc-region of IgG.
  • IgG and FcRn The interaction between IgG and FcRn is strictly pH dependent and occurs in a 1:2 stoichiometry, with one IgG binding to two FcRn molecules via its two heavy chains ( Huber, A.H., et al., J. Mol. Biol. 230 (1993) 1077-1083 ).
  • FcRn binding occurs in the endosome at acidic pH (pH ⁇ 6.5) and IgG is released at the neutral cell surface (pH of about 7.4).
  • the pH-sensitive nature of the interaction facilitates the FcRn-mediated protection of IgGs pinocytosed into cells from intracellular degradation by binding to the receptor within the acidic environment of endosomes.
  • FcRn then facilitates the recycling of IgG to the cell surface and subsequent release into the blood stream upon exposure of the FcRn-IgG complex to the neutral pH environment outside the cell.
  • FcRn binding portion of an Fc-region denotes the part of an antibody heavy chain polypeptide that extends approximately from EU position 243 to EU position 261 and approximately from EU position 275 to EU position 293 and approximately from EU position 302 to EU position 319 and approximately from EU position 336 to EU position 348 and approximately from EU position 367 to EU position 393 and EU position 408 and approximately from EU position 424 to EU position 440.
  • one or more of the following amino acid residues according to the EU numbering of Kabat are altered F243, P244, P245, K246, P247, K248, D249, T250, L251, M252, I253, S254, R255, T256, P257, E258, V259, T260, C261, F275, N276, W277, Y278, V279, D280, V282, E283, V284, H285, N286, A287, K288, T289, K290, P291, R292, E293, V302, V303, S304, V305, L306, T307, V308, L309, H310, Q311, D312, W313, L314, N315, G316, K317, E318, Y319, I336, S337, K338, A339, K340, G341, Q342, P343, R344, E345, P346, Q347, V348, C367, V369, F37
  • full length antibody denotes an antibody having a structure substantially similar to a native antibody structure.
  • a full length antibody comprises two full length antibody light chains comprising a light chain variable domain and a light chain constant domain and two full length antibody heavy chains comprising a heavy chain variable domain, a first constant domain, a hinge region, a second constant domain and a third constant domain.
  • a full length antibody may comprise further domains, such as e.g. additional scFv or a scFab conjugated to one or more of the chains of the full length antibody. These conjugates are also encompassed by the term full length antibody.
  • host cell refers to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells.
  • Host cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.
  • derived from denotes that an amino acid sequence is derived from a parent amino acid sequence by introducing alterations at at least one position.
  • a derived amino acid sequence differs from the corresponding parent amino acid sequence at at least one corresponding position (numbering according to Kabat EU index for antibody Fc-regions).
  • an amino acid sequence derived from a parent amino acid sequence differs by one to fifteen amino acid residues at corresponding positions.
  • an amino acid sequence derived from a parent amino acid sequence differs by one to ten amino acid residues at corresponding positions.
  • an amino acid sequence derived from a parent amino acid sequence differs by one to six amino acid residues at corresponding positions.
  • a derived amino acid sequence has a high amino acid sequence identity to its parent amino acid sequence.
  • an amino acid sequence derived from a parent amino acid sequence has 80 % or more amino acid sequence identity.
  • an amino acid sequence derived from a parent amino acid sequence has 90 % or more amino acid sequence identity.
  • an amino acid sequence derived from a parent amino acid sequence has 95 % or more amino acid sequence identity.
  • human Fc-region polypeptide denotes an amino acid sequence which is identical to a "native” or “wild-type” human Fc-region polypeptide.
  • variant (human) Fc-region polypeptide denotes an amino acid sequence which derived from a "native” or "wild-type” human Fc-region polypeptide by virtue of at least one "amino acid alteration".
  • a "human Fc-region” is consisting of two human Fc-region polypeptides.
  • a “variant (human) Fc-region” is consisting of two Fc-region polypeptides, whereby both can be variant (human) Fc-region polypeptides or one is a human Fc-region polypeptide and the other is a variant (human) Fc-region polypeptide.
  • a “humanized” antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., the CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.
  • a humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody.
  • a "humanized form" of an antibody, e.g., a non-human antibody refers to an antibody that has undergone humanization.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • polyclonal antibody preparations typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier "monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • Native antibodies refer to naturally occurring immunoglobulin molecules with varying structures.
  • native IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. From N- to C-terminus, each heavy chain has a variable region (VH), also called a variable heavy domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3).
  • VH variable region
  • VL variable region
  • the light chain of an antibody may be assigned to one of two types, called kappa ( ⁇ ) and lambda ( ⁇ ), based on the amino acid sequence of its constant domain.
  • a “pharmaceutically acceptable carrier” refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • plasmid refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked.
  • the term includes the plasmid as a self-replicating nucleic acid structure as well as the plasmid incorporated into the genome of a host cell into which it has been introduced.
  • Certain plasmids are capable of directing the expression of nucleic acids to which they are operatively linked. Such plasmids are referred to herein as "expression plasmid”.
  • positive linear pH gradient denotes a pH gradient starting at a low (i.e. more acidic) pH value and ending at a higher (i.e. less acidic, neutral or alkaline) pH value.
  • the positive linear pH gradient starts at a pH value of about 5.5 and ends at a pH value of about 8.8.
  • recombinant antibody denotes all antibodies (chimeric, humanized and human) that are prepared, expressed, created or isolated by recombinant means. This includes antibodies isolated from a host cell such as a NS0, HEK, BHK or CHO cell or from an animal (e.g. a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression plasmid transfected into a host cell. Such recombinant antibodies have variable and constant regions in a rearranged form.
  • the recombinant antibodies as reported herein can be subjected to in vivo somatic hypermutation.
  • the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germ line VH and VL sequences, may not naturally exist within the human antibody germ line repertoire in vivo.
  • a “solid phase” denotes a non-fluid substance, and includes particles (including microparticles and beads) made from materials such as polymer, metal (paramagnetic, ferromagnetic particles), glass, and ceramic; gel substances such as silica, alumina, and polymer gels; capillaries, which may be made of polymer, metal, glass, and/or ceramic; zeolites and other porous substances; electrodes; microtiter plates; solid strips; and cuvettes, tubes or other spectrometer sample containers.
  • a solid phase component of an assay is distinguished from inert solid surfaces in that a "solid support" contains at least one moiety on its surface, which is intended to interact chemically with a molecule.
  • a solid phase may be a stationary component, such as a chip, tube, strip, cuvette, or microtiter plate, or may be non-stationary components, such as beads and microparticles.
  • Microparticles can also be used as a solid support for homogeneous assay formats.
  • Such particles include polymer particles such as polystyrene and poly (methylmethacrylate); gold particles such as gold nanoparticles and gold colloids; and ceramic particles such as silica, glass, and metal oxide particles. See for example Martin, C.R., et al., Analytical Chemistry-News & Features, May 1 (1998) 322A-327A , which is incorporated herein by reference.
  • the solid support is sepharose.
  • bivalent as used within the current application denotes the presence of a specified number of binding sites in a (antibody) molecule.
  • bivalent tetravalent
  • hexavalent denote the presence of two binding site, four binding sites, and six binding sites, respectively, in a (antibody) molecule.
  • the bispecific antibodies as reported herein as reported herein are in one preferred embodiment "bivalent”.
  • variable region or “variable domain” refer to the domain of an antibody heavy or light chain that is involved in binding of the antibody to its antigen.
  • the variable domains of the heavy chain and light chain (VH and VL, respectively) of an antibody generally have similar structures, with each domain comprising four framework regions (FRs) and three hypervariable regions (HVRs) (see, e.g., Kindt, T.J. et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., N.Y. (2007), page 91 ).
  • a single VH or VL domain may be sufficient to confer antigen-binding specificity.
  • antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano, S. et al., J. Immunol. 150 (1993) 880-887 ; Clackson, T. et al., Nature 352 (1991) 624-628 ).
  • beta-2-microglobulin is from the same species as the FcRn.
  • the soluble extracellular domain of FcRn (SEQ ID NO: 31 for human FcRn) with C-terminal His-Avi Tag (SEQ ID NO: 32) was co-expressed with ⁇ 2 -microglobulin (SEQ ID NO: 33 for human beta-2-microglobulin) in mammalian cells.
  • the non-covalent FcRn-microglobulin complex was biotinylated and loaded onto streptavidin derivatized sepharose.
  • variants of antibody no. 5 were synthesized covering the biophysical space of charges and hydrophobicities normally seen in antibody Fvs.
  • Variants carrying positively charged patches showed strong heparin and FcRn column retention (relative retention of the FcRn column of 0.5 and more and relative retention on the heparin column of 0.8 or more), which predicts fast clearance.
  • Variants carrying negatively charged patches showed weak heparin and FcRn column retention (relative retention of the FcRn column of 0.25 and less and relative retention on the heparin column of 0.6 or less), which predicts slow clearance.
  • the resulting antibody variant behaves as if it was carrying only positively charged patches.
  • the modified antibody has at least one additional positively charged patch on its (solvent-exposed) surface.
  • the modified antibody has the same (surface) net charge as the parent antibody.
  • a (permanently) negatively charged amino acid residue is selected from the group consisting of glutamate and aspartate.
  • a (permanently) positively charged amino acid residue is selected from the group consisting of arginine and lysine.
  • the pH-dependently charged amino acid residue is histidine.
  • a pH-dependently charged amino acid residue has a first (net) charge at pH 6 and an opposite second (net) charge at pH 8.
  • Fc-region mutations to increase FcRn affinity should improve the pharmacokinetic properties of antibodies by raising FcRn recycling efficiency (increased binding, important at pH 6.0) and should shift antibodies from the left side of Figure 12 into the peak area.
  • variant FcRn rel. Retention [min] (-wt) CL (ml/kg/day) Tg32 hFcRn +/+ mice fold CL change (to WT) in Tg32 hFcRn +/+ 1-WT 0 5.26 1 1-YTE 3.72 3.69 0.70 104-WT 0 3.67 1 104-YTE 3.6 3.28 0.89 108-WT 0 5.26 1 108-YTE 3.63 4.53 0.86 2-WT 0 3.63 1 2-YTE 3.64 2.72 0.75 20-WT 0 4.02 1 20-H433K/N434F 7.74 6.46 1.61 20-M428L 1.52 2.9 0.72 20-T307H/N434H 3.75 3.21 0.8 20-YTE 3.48 3.03 0.75 27-WT 0 30.1 1 27-Y436A -
  • Mutation Y436A results in a reduced FcRn binding and thus, as expected the clearance is increased (Y-axis value above 1).
  • Mutations H433K/N434F results in a dramatically increased FcRn binding at pH 7.4 (X-axis value or more than 5, i.e. dissociation from FcRn at highly elevated pH value), which, as expected, results in an increased clearance (Y-axis value above 1) as dissociation of the antibody from FcRn at pH 7.4 is impaired and thereby FcRn-mediated recycling is reduced.
  • one aspect of the current invention is a method for providing an antibody with improved in vivo half-live, comprising the following steps:
  • the FcRn is a non-covalent complex with human beta-2-microglobulin (b2m).
  • the method comprises as step b) the following step: b) modifying the Fc-region of the antibody by introducing mutations that increase the binding of the Fc-region to human FcRn to obtain a modified antibody, and modifying the Fv of the antibody by
  • a relative retention time region comprising predominantly antibodies with slow clearance is defined. This region is defined by a relative retention time on the FcRn affinity chromatography column of less than 1.78 (with an oxidized (H2O2-treated) anti-Her3 antibody preparation as reference antibody) and by a relative retention time on the heparin affinity chromatography column of less than 0.87 (with an anti-pTau antibody as reference antibody).
  • the modified antibody has at least one additional negatively charged patch on its (solvent-exposed) surface.
  • the modified antibody has the same (surface) net charge as the parent antibody.
  • a (permanently) negatively charged amino acid residue is selected from the group consisting of glutamate and aspartate.
  • a (permanently) positively charged amino acid residue is selected from the group consisting of arginine and lysine.
  • the pH-dependently charged amino acid residue is histidine.
  • a permanently charged amino acid residue has the same (net) charge in the pH range from pH 6 to pH 8.
  • a pH-dependently charged amino acid residue has a first (net) charge at pH 6 and an opposite second (net) charge at pH 8.
  • the standard sample is an oxidized antibody preparation comprising the reference antibody with respect to the methionine residues at position 252 in the heavy chain CH2 domains in non-oxidized form, in mono-oxidized form (only one of the two methionins at position 252 is oxidized) and in bi-oxidized form (both methionine residues at position 252 are oxidized) (numbering according to Kabat).
  • the first reference antibody is an anti-Her3 antibody that has a heavy chain with the amino acid sequence of SEQ ID NO: 03 and a light chain with the amino acid sequence of SEQ ID NO: 04.
  • the first threshold value in step f) is 2. In one embodiment the first threshold value is 1.8. In one embodiment the first threshold value is 1.78.
  • the second reference antibody in step f) is an anti-pTau antibody that has a heavy chain with the amino acid sequence of SEQ ID NO: 01 and a light chain with the amino acid sequence of SEQ ID NO: 02.
  • the second threshold value is 1. In one embodiment the second threshold value is 0.8. In one embodiment the second threshold value is 0.78.
  • step f) is providing an antibody with improved in vivo half-live if the modified antibody has
  • step e affinity chromatography of step e) with a positive linear pH gradient is used an immobilized non-covalent complex of a neonatal Fc receptor (FcRn) and beta-2-microglobulin (b2m) as affinity chromatography ligand,
  • FcRn neonatal Fc receptor
  • b2m beta-2-microglobulin
  • the pH gradient of step e) is from a first pH value to a second pH value whereby the first pH value is pH 5.5 and the second pH value is pH 8.8.
  • beta-2-microglobulin is from the same species as the FcRn.
  • the FcRn is selected from human FcRn, cynomolgus FcRn, mouse FcRn, rat FcRn, sheep FcRn, dog FcRn, pig FcRn, minipig FcRn, and rabbit FcRn.
  • beta-2-microglobulin is from the same species as the FcRn.
  • beta-2-microglobulin is from a different species as the FcRn.
  • the soluble extracellular domain of FcRn (SEQ ID NO: 31 for human FcRn) with C-terminal His-Avi Tag (SEQ ID NO: 32) was co-expressed with ⁇ 2 -microglobulin (SEQ ID NO: 33 for human beta-2-microglobulin) in mammalian cells.
  • the non-covalent FcRn-microglobulin complex was biotinylated and loaded onto streptavidin derivatized sepharose.
  • the reference antibody for the FcRn affinity chromatography is the anti-HER3 antibody with SEQ ID NO: 03 (heavy chain) and SEQ ID NO: 04 (light chain).
  • the reference antibody for the heparin affinity chromatography is the anti-pTau antibody with SEQ ID NO: 01 (heavy chain) and SEQ ID NO: 02 (light chain).
  • the antibody is a monospecific antibody or antibody fragment of fusion polypeptide, or a bispecific antibody or antibody fragment of fusion polypeptide, or a trispecific antibody or antibody fragment of fusion polypeptide, or a tetraspecific antibody or antibody fragment of fusion polypeptide.
  • the antibody is an antibody of the class IgG. In one embodiment the antibody is an antibody of the subclass IgG1, IgG2, IgG3 or IgG4. In one embodiment the antibody is an antibody of the subclass IgG1 or IgG4.
  • the neonatal Fc-receptor (FcRn) is important for the metabolic fate of antibodies of the IgG class in vivo.
  • the FcRn functions to salvage wild-type IgG from the lysosomal degradation pathway, resulting in reduced clearance and increased half-life. It is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex-like protein ( ⁇ -FcRn) and a 15 kDa ⁇ 2-microglobulin ( ⁇ 2m).
  • ⁇ -FcRn major histocompatibility complex-like protein
  • ⁇ 2m microglobulin
  • an antibody of the class IgG and the FcRn is pH dependent and occurs in a 1:2 stoichiometry, i.e. one IgG antibody molecule can interact with two FcRn molecules via its two heavy chain Fc-region polypeptides (see e.g. Huber, A.H., et al., J. Mol. Biol. 230 (1993) 1077-1083 ).
  • an IgGs in vitro FcRn binding properties/characteristics are indicative of its in vivo pharmacokinetic properties in the blood circulation.
  • the amino acid residues interacting with the FcRn are located approximately between EU position 243 and EU position 261, approximately between EU position 275 and EU position 293, approximately between EU position 302 and EU position 319, approximately between EU position 336 and EU position 348, approximately between EU position 367 and EU position 393, at EU position 408, and approximately between EU position 424 and EU position 440.
  • antibodies with reduced half-life in the blood circulation are desired.
  • drugs for intravitreal application should have a long half-live in the eye and a short half-life in the blood circulation of the patient.
  • Such antibodies also have the advantage of increased exposure to a disease site, e.g. in the eye.
  • Fc-region residues critical to the mouse Fc-region- -mouse FcRn interaction have been identified by site-directed mutagenesis (see e.g. Dall'Acqua, W.F., et al. J. Immunol 169 (2002) 5171-5180 ).
  • Residues I253, H310, H433, N434, and H435 are involved in the interaction ( Medesan, C., et al., Eur. J. Immunol. 26 (1996) 2533-2536 ; Firan, M., et al., Int. Immunol.
  • Residues I253, H310, and H435 were found to be critical for the interaction of human Fc with murine FcRn ( Kim, J.K., et al., Eur. J. Immunol. 29 (1999) 2819-2855 ).
  • Residues M252Y, S254T, T256E have been described by Dall' Acqua et al. to improve FcRn binding by protein-protein interaction studies ( Dall'Acqua, W.F., et al. J. Biol. Chem. 281 (2006) 23514-23524 ).
  • P257I/Q311I humanized IgG1
  • P257I/Q311I increased at pH 6 (human, Cynomolgus, mouse) reduced in mice P257I/N434H reduced in Cynomolgus Datta-Mannan, A., et al., Drug Metab. Dispos.
  • the biochemical characterization included size exclusion chromatography (Waters BioSuite TM 250 7.8 x 300 mm, eluent: 200 mM KH 2 PO 4 , 250 mM KCl, pH 7.0) and analysis of the molecular weight distribution using the BioAnalyzer 2100 (Agilent technologies, Santa Clara, CA, USA).
  • variants of expression plasmids for transient expression e.g. in HEK293-F cells based either on a cDNA organization with or without a CMV-Intron A promoter or on a genomic organization with a CMV promoter were applied.
  • the transcription unit of the antibody gene was composed of the following elements:
  • the fusion genes comprising the antibody chains were generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques by connection of the according nucleic acid segments e.g. using unique restriction sites in the respective plasmids.
  • the subcloned nucleic acid sequences were verified by DNA sequencing.
  • larger quantities of the plasmids were prepared by plasmid preparation from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel).
  • the antibodies were generated by transient transfection with the respective plasmids (e.g. encoding the heavy chain, as well as the corresponding light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instruction. Briefly, HEK293-F cells (Invitrogen) growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle TM 293 expression medium (Invitrogen) were transfected with a mix of the respective expression plasmids and 293fectin TM or fectin (Invitrogen).
  • HEK293-F cells Invitrogen growing in suspension either in a shake flask or in a stirred fermenter in serum-free FreeStyle TM 293 expression medium (Invitrogen) were transfected with a mix of the respective expression plasmids and 293fectin TM or fectin (Invitrogen).
  • HEK293-F cells were seeded at a density of 1*10 6 cells/mL in 600 mL and incubated at 120 rpm, 8 % CO 2 .
  • the antibodies were purified from cell culture supernatants by affinity chromatography using MabSelectSure-Sepharose TM (GE Healthcare, Sweden), hydrophobic interaction chromatography using butyl-Sepharose (GE Healthcare, Sweden) and Superdex 200 size exclusion (GE Healthcare, Sweden) chromatography.
  • sterile filtered cell culture supernatants were captured on a MabSelectSuRe resin equilibrated with PBS buffer (10 mM Na 2 HPO 4 , 1 mM KH 2 PO 4 , 137 mM NaCl and 2.7 mM KCl, pH 7.4), washed with equilibration buffer and eluted with 25 mM sodium citrate at pH 3.0.
  • the eluted antibody fractions were pooled and neutralized with 2 M Tris, pH 9.0.
  • the antibody pools were prepared for hydrophobic interaction chromatography by adding 1.6 M ammonium sulfate solution to a final concentration of 0.8 M ammonium sulfate and the pH adjusted to pH 5.0 using acetic acid.
  • the antibodies were applied to the resin, washed with equilibration buffer and eluted with a linear gradient to 35 mM sodium acetate pH 5.0.
  • the antibody containing fractions were pooled and further purified by size exclusion chromatography using a Superdex 200 26/60 GL (GE Healthcare, Sweden) column equilibrated with 20 mM histidine, 140 mM NaCl, pH 6.0.
  • the antibody containing fractions were pooled, concentrated to the required concentration using Vivaspin ultrafiltration devices (Sartorius Stedim Biotech S.A., France) and stored at -80 °C.
  • the pharmacokinetic parameters were calculated by non-compartmental analysis using WinNonlin TM 1.1.1 (Pharsight, CA, USA).
  • Plasma clearance was calculated as Dose rate (D) divided by AUC 0-inf .
  • the biotinylated FcRn was dialyzed against 20 mM MES buffer comprising 140 mM NaCl, pH 5.5 (buffer A) at 4°C overnight to remove excess of biotin.
  • t rel , i t i t pTau ( t rel,i : relative retention time of peak i; t i : retention time of peak i; t pau : retention time of the anti-pTau antibody peak).
  • mutants were prepared by site directed mutagenesis of one parent antibody V1 in the Fab: V2, V20, V27, V104 and V108.
  • mice lacking the mFcRn ⁇ -chain B6.129X1-Fcgrttm1Dcr/DcrJ; abbreviated FcRn-ko
  • FcRn-ko homozygous B6.Cg-Fcgrt ⁇ tm1Dcr[Tg(FCGRT)32Dcr/DcrJ mice
  • hFcRn Tg32 mice lacking the mFcRn ⁇ -chain
  • FcRn-ko homozygous B6.Cg-Fcgrt ⁇ tm1Dcr[Tg(FCGRT)32Dcr/DcrJ mice
  • Serial blood samples were collected over 4 weeks or 96 h in hFcRn Tg32 and FcRn-k
  • the concentrations of the human therapeutic antibodies in murine serum samples were determined using an electro-chemiluminescence immunoassay (ECLIA) method specific for the human Fab moiety of the administered antibody and its variants. Briefly, samples, diluted with assay buffer, were incubated with capture and detection molecules for 9 min at 37 °C. Biotinylated mAb ⁇ H-Fab(kappa)>M-IgG-Bi was used as capture molecule and a ruthenium(II)tris(bipyridyl)32+ labeled mAb ⁇ H-Fab(CH1)>M-1.19.31-1gG-S-Ru mouse monoclonal antibody was used for detection.
  • ELIA electro-chemiluminescence immunoassay
  • Streptavidin-coated magnetic microparticles were added and incubated for additional 9 min at 37 °C to allow complex formation due to biotin-streptavidin interactions. Complexes were magnetically captured on an electrode and a chemiluminescent signal generated using the co-reactant tripropylamine (TPA) was measured by a photomultiplier detector. All serum samples and positive or negative control samples were analyzed in replicates and calibrated against the corresponding antibody that was administered.
  • TPA co-reactant tripropylamine
  • Analytical FcRn affinity chromatography was performed using a commercially available FcRn affinity column (Part.No. 08128057001, Roche Diagnostic, Mannheim Germany), pre-equilibrated with 80% buffer A (20 mM MES sodium salt, 140 mM NaCl, pH 5.5) and 20% buffer B (20 mM Tris/HCl, 140 mM NaCl, pH 8.8) at a flow rate of 0.5 mL/min and a column temperature of 25 °C. Samples were prepared as above. A total of 30 ⁇ g protein was injected. Ten minutes post injection, a linear gradient from 20 - 100% buffer B over 70 minutes was started.

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Claims (14)

  1. Verfahren zum Bereitstellen eines Antikörpers mit verbesserter In-vivo-Halbwertszeit, umfassend die folgenden Schritte:
    a) Bestimmen der relativen Retentionszeit eines parentalen Antikörpers in einer FcRn-Affinitätschromatographie,
    b) Modifizieren der Fc-Region des Antikörpers durch Einbringen von Mutationen, die das Binden der Fc-Region an humanen FcRn verstärken, unter Erhalt eines modifizierten Antikörpers,
    c) Bestimmen der relativen Retentionszeit des modifizierten Antikörpers in einer FcRn-Affinitätschromatographie,
    d) Wiederholen von Schritt b), wenn die Änderung der relativen Retentionszeit zwischen dem parentalen Antikörper und dem modifizierten Antikörper weniger als 1 Minute und mehr als 5 Minuten beträgt, wobei im Falle, dass die Änderung der relativen Retentionszeit weniger als 1 Minute beträgt, Mutationen gewählt werden, die zu einem weiter verstärkten Binden der Fc-Region an humanen FcRn führen, oder im Falle, dass die Änderung der relativen Retentionszeit mehr als 5 Minuten beträgt, Mutationen gewählt werden, die zu einem weniger verstärkten Binden der Fc-Region an humanen FcRn führen,
    e) Bereitstellen eines Antikörpers mit verbesserter In-vivo-Halbwertszeit, wenn die Änderung der relativen Retentionszeit zwischen dem parentalen Antikörper und dem modifizierten Antikörper mehr als 1 Minute und weniger als 5 Minuten beträgt.
  2. Verfahren nach Anspruch 1, wobei die Änderung der relativen Retentionszeit zwischen dem parentalen Antikörper und dem modifizierten Antikörper mehr als etwa 2 Minuten und weniger als etwa 4,5 Minuten beträgt.
  3. Verfahren nach einem der Ansprüche 1 bis 2, wobei die Änderung der relativen Retentionszeit zwischen dem parentalen Antikörper und dem modifizierten Antikörper mehr als etwa 3 Minuten und weniger als etwa 4 Minuten beträgt.
  4. Verfahren nach einem der Ansprüche 1 bis 3, wobei die FcRn-Affinitätschromatographie wie folgt durchgeführt wird:
    - eine FcRn-Affinitätschromatographiesäule, umfassend etwa 1 ml einer Streptavidin-Agarose-Matrix mit daran über nicht kovalente Biotin-Streptavidin-Interaktion konjugiertem humanem FcRn, wird mit 80 Vol.-% Puffer A, umfassend 20 mM MES-Natriumsalz, 140 mM NaCl, pH 5,5, und 20 Vol.-% Puffer B, umfassend 20 mM Tris/HCl, 140 mM NaCl, pH 8,8, bei einer Fließgeschwindigkeit von 0,5 ml/min und einer Säulentemperatur von 25 °C äquilibriert;
    - insgesamt 30 mg des Antikörpers werden in demselben Gemisch aus 80 Vol.-% Puffer A und 20 Vol.-% Puffer B hergestellt und auf die äquilibrierte Säule injiziert;
    - zehn Minuten nach der Injektion wird ein linearer Gradient von 20 Vol.-% bis 100 Vol.-% Puffer B über 70 Minuten gestartet;
    - gegebenenfalls werden 100 Vol.-% Puffer B für 10 Minuten gehalten, bevor die Säule erneut mit 80 Vol.-% Puffer A und 20 Vol.-% Puffer B äquilibriert wird;
    wobei der Nachweis mit einem auf 280 nm eingestellten UV-Detektor durchgeführt wird;
    wobei eine relative Retentionszeit auf der FcRn-Affinitätschromatographiesäule gemäß der folgenden Gleichung berechnet wird: wobei trel,i: relative Retentionszeit von Peak i; ti: Retentionszeit von Peak i; tpeak2: Retentionszeit des zweiten Peaks eines teilweise oxidierten Anti-Her3-Antikörpers; tPeak3: Retentionszeit des dritten Peaks eines teilweise oxidierten Anti-Her3-Antikörpers auf einer FcRn-Affinitätschromatographiesäule mit einem wie vorstehend im Anspruch aufgeführten Gradienten.
  5. Verfahren nach einem der Ansprüche 1 bis 4, wobei das Verfahren als Schritt b) den folgenden Schritt umfasst:
    b) Modifizieren der Fc-Region des Antikörpers durch Einbringen von Mutationen, die das Binden der Fc-Region an humanen FcRn verstärken, unter Erhalt eines modifizierten Antikörpers
    und
    Modifizieren des Fv des Antikörpers durch
    - Verringern der Größe positiv geladener Bereiche,
    - Verringern der Größe positiv geladener Bereiche und Erhöhen der Größe negativ geladener Bereiche,
    - gleichmäßiges Verteilen der Gesamtladungen in dem Fv oder Fab.
  6. Verfahren nach Anspruch 5, wobei die Ladungsverteilung in dem Fv-Fragment durch Folgendes geändert wird
    i) Ändern mindestens eines negativ geladenen oder nicht geladenen Aminosäurerestes in einen positiv geladenen Aminosäurerest oder
    ii) Ändern mindestens eines positiv geladenen oder nicht geladenen Aminosäurerestes in einen negativ geladenen Aminosäurerest oder
    iii) Ändern mindestens eines geladenen Aminosäurerestes in einen Aminosäurerest mit entgegengesetzter Ladung oder
    iv) Ändern mindestens eines dauerhaft geladenen Aminosäurerestes in einen pH-abhängig geladenen Aminosäurerest.
  7. Verfahren nach einem der Ansprüche 1 bis 6, wobei das Verfahren ferner die folgenden Schritte umfasst:
    e) Durchführen einer FcRn-Affinitätschromatographie mit einem positiven linearen pH-Gradienten und einer Heparinaffinitätschromatographie mit einem positiven linearen Leit-/Salzgradienten mit dem parentalen Antikörper und dem modifizierten Antikörper,
    und
    f) Bereitstellen eines Antikörpers mit verbesserter In-vivo-Halbwertszeit, wenn der modifizierte Antikörper Folgendes aufweist
    i) eine relative Retentionszeit auf der FcRn-Affinitätschromatographiesäule, die unter denselben Elutionsbedingungen weniger als die Retentionszeit des parentalen Antikörpers auf derselben FcRn-Affinitätschromatographiesäule beträgt, oder
    ii) eine relative Retentionszeit auf der Heparinaffinitätschromatographiesäule, die unter denselben Elutionsbedingungen weniger als die Retentionszeit des parentalen Antikörpers auf derselben Heparinaffinitätschromatographiesäule beträgt, oder
    iii) sowohl i) als auch ii).
  8. Verfahren nach einem der Ansprüche 5 bis 7, wobei der modifizierte Antikörper mindestens einen zusätzlichen negativ geladenen Bereich auf seiner lösungsmittelexponierten Oberfläche aufweist.
  9. Verfahren nach einem der Ansprüche 5 bis 8, wobei der modifizierte Antikörper dieselbe Oberflächennettoladung wie der parentale Antikörper aufweist.
  10. Verfahren nach einem der Ansprüche 5 bis 9, wobei ein negativ geladener Aminosäurerest aus der Gruppe ausgewählt ist, die aus Glutamat und Aspartat besteht.
  11. Verfahren nach einem der Ansprüche 5 bis 10, wobei ein positiv geladener Aminosäurerest aus der Gruppe ausgewählt ist, die aus Arginin und Lysin besteht.
  12. Verfahren nach einem der Ansprüche 5 bis 11, wobei der pH-abhängig geladene Aminosäurerest Histidin ist.
  13. Verfahren nach einem der Ansprüche 5 bis 12, wobei ein dauerhaft geladener Aminosäurerest im pH-Bereich von pH 6 bis pH 8 dieselbe Nettoladung aufweist.
  14. Verfahren nach einem der Ansprüche 5 bis 7, wobei ein pH-abhängig geladener Aminosäurerest bei pH 6 eine erste Nettoladung und bei pH 8 eine entgegengesetzte zweite Nettoladung aufweist.
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JP2022512798A (ja) 2022-02-07
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JP7622134B2 (ja) 2025-01-27
WO2020084032A1 (en) 2020-04-30
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